1. Technical Field
The invention relates to phenylalanine-containing powders, particularly spray-dried powders, which contain at least phenylalanine and a protein, the protein preferably being an active substance and particularly a pharmaceutical active substance. The inventive powders contain a phenylalanine fraction of at least 30% (w/w), preferably 40% (w/w) and optionally at least one second pharmaceutically acceptable excipient, namely a sugar, which enhances the protein stability. The invention further relates to a process for preparing these phenylalanine-containing powders as well as the use thereof particularly as inhalative pharmaceutical compositions. Preferred proteins are pharmaceutical active substances such as antibodies, parts of antibodies, fusion proteins with antibodies or parts of antibodies, hormones, growth factors, enzymes, cytokines, interferons or the like for local treatment of the airways or for systemic treatment.
2. Background
Protein preparations or active substances/active substance preparations formulated in aqueous solutions are in some cases prone to instability which may lead to reduced efficacy or bioactivity and increased toxicity or incompatibilities. This applies both to conventional pharmaceuticals and to proteins and particularly active substances containing peptides or proteins. The stability of proteins or pharmaceutical active substances may be favourably influenced by altering the structure (internal) or by adding suitable excipients (external).
A conventional method of externally stabilising proteins or pharmaceutical active substances is the use of suitable excipients. Excipients may be divided roughly into the following categories: sugars and polyols, amino acids, amines, salts, polymers and surfactants.
Sugars and polyols are frequently used as non-specific stabilisers. Their stabilising effect in proteins or biological active substances is predominantly put down to “preferential exclusion” (Xie and Timasheff, 1997, Biophysical Chemistry, 64(1-3), 25-43; Xie and Timasheff, 1997, Protein Science, 6(1), 211-221; Timasheff, 1998, Advances in protein chemistry, 51, 355-432). When choosing sugars, reducing sugars are usually avoided in the case of proteins or biological active substances. Saccharose and trehalose, being non-reducing sugars, are preferably used. Further examples of suitable excipients are glucose, sorbitol, glycerol (Boctor and Mehta, 1992, Journal of Pharmacy and Pharmacology, 44 (7), 600-3; Timasheff, 1993, Annual review of biophysics and biomolecular structure, 22, 67-97; Chang et al., 1993, Pharmaceutical Research, 10(10), 1478-83) and mannitol (Hermann et al., 1996, Pharmaceutical Biotechnology, 9 (Formulation, Characterization, and Stability of protein Drugs) 303-328; Chan et al., 1996, Pharmaceutical Research, 13(5), 756-761). It is also known that all kinds of polymers have a stabilising effect on proteins or pharmaceutical active substances such as for example antibodies. Human serum albumin (HAS) which has frequently been used in the past does indeed have very good stabilising properties but because of its potential contamination with “blood-borne” pathogens it is unsuitable in the mean time. Of the polymers known hitherto, hydroxypropyl-β-cyclodextrin (HP-β-CD) has proved particularly suitable, as it can also be safely administered parenterally. Other examples are higher-molecular dextrans (18 to 82 kD), polyvinylpyrrolidones (PVP), heparin, type A and B gelatine as well as hydroxyethyl-starch (HES), heparin, dextran sulphate, polyphosphoric acid, poly-L-glutamic acid, poly-L-lysine.
In addition to sugars and polyols, amino acids may also be used as stabilisers, on their own or in conjunction with other excipients. Preferably amino acids are used in the stabilisation of proteins. For example the addition of histidine, glycine, sodium-aspartate (Na-Asp), glutamate and lysine hydrochloride (Lys-HCl) inhibits the aggregation of rhKGF in 10 mM sodium phosphate buffer (pH 7.0) together with 5% mannitol (Zhang et al., 1995, Biochemistry, 34 (27), 8631-41). The combination of amino acids and propyleneglycol improves for example the structural stability of rhCNTF (Dix et al, 1995, Pharmaceutical Research (Supplement), 12, S97). Lysine and arginine increase the heat stability of IL-1R (Tm increase), whereas glycine and alanine have a destabilising effect (Remmele et al., 1998, Pharmaceutical Research, 15(2), 200-208).
Moreover, the stability of powders containing protein or pharmaceutical active substances can be increased by various drying processes. The drying is usually carried out in the presence of excipients which should maintain the stability of the proteins or active substances and improve the properties of the dry powders. A crucial factor in stabilising by drying is the immobilisation of the protein or active substance in an amorphous matrix. The amorphous state has high viscosity with low molecular mobility and low reactivity. Advantageous excipients must therefore be capable of forming an amorphous matrix with the highest possible glass transition temperature in which the protein or active substance is embedded. The choice of excipients thus depends particularly on their stabilising qualities. In addition, however, factors such as the pharmaceutical acceptance of the excipient and its influence on particle formation, dispersibility and flow properties play a decisive role, particularly in spray-drying processes.
Spray-drying is a particularly suitable process for increasing the chemical and physical stability of proteins or pharmaceutical active substances of the peptide/protein type (cf. Maa et al., 1998, Pharmaceutical Research, 15(5), 768-775). Particularly in the field of pulmonary treatment spray drying is increasingly used (U.S. Pat. No. 5,626,874; U.S. Pat. No. 5,972,388; Broadhead et al., 1994, J. Pharm Pharmacol., 46(6), 458-467), as administration by inhalation is now an alternative in the treatment of systemic diseases (WO 99/07340). The prerequisite for this is that the mean aerodynamic particle size (MMAD=mass median aerodynamic diameter) of the powder particles is in the range from 1-10 μm, preferably 1-7.5 μm, so that the particles can penetrate deep into the lungs and thus enter the bloodstream. DE-A-179 22 07, for example, describes the preparation of corresponding spray dried particles. In the meantime a number of methods of producing corresponding powders have been described (WO 95/31479; WO 96/09814; WO 96/32096; WO 96/32149; WO 97/41833; WO 97/44013; WO 98/16205; WO 98/31346; WO 99/66903; WO 00/10541; WO 01/13893; Maa et al., 1998, supra; Vidgrén et al., 1987, Int. J. Pharmaceutics, 35, 139-144; Niven et al., 1994, Pharmaceutical Research, 11(8), 1101-1109).
Sugar and alcohols thereof such as, for example, trehalose, lactose, saccharose or mannitol and various polymers have proved suitable as excipients (Maa et al., 1997, Pharm. Development and Technology, 2(3), 213-223; Maa et al., 1998, supra; Dissertation Adler, 1998, University of Erlangen; Costantino, et al., 1998, J. Pharm. Sci., 87(11), 1406-1411).
However, the excipients predominantly used have various drawbacks. The addition of trehalose and mannitol, for example, impairs the flow properties of spray-drying formulations (C. Bosquillon et al., 2001 Journal of Controlled Release, 70(3), 329-339). Spray-dried trehalose often causes serious sticking of the resulting particles (L. Mao et. al, 2004 Respiratory Drug Delivery IX, S. 653-656). This is associated with technical processing problems connected with the yields of powder and the robustness of the process, as well as a deterioration in the bioavailability of the powder for pulmonary application, caused by a reduction in the fine particle fraction that can be obtained. Moreover, mannitol has a tendency to recrystallise in amounts of more than 20 percent by weight (Costantino et al., 1998, supra), as a result of which its stabilising effects are dramatically reduced. Lactose, a frequently used excipient, does improve the flow properties of spray-drying formulations (C. Bosquillon et al., 2001, supra), but is problematic particularly in the formulation of proteins or peptide/protein-containing active substances, as lactose can enter into destabilising Maillard reactions with peptides/proteins as a result of its reducing property.
Besides protein stabilisation using excipients, however, optimising the physicochemical properties of spray-dried powders is the focus of the recipe development. In particular, powders, particularly spray-dried powders, have a tendency to cohesive and adhesive characteristics. One important reason for this is the particles size of <10 μm which is necessary for pulmonary administration. At these small particle sizes, particle interactions such as e.g. Van-der-Waals forces, capillary forces, dipolar interactions and electrostatic interactions, predominate over gravitational forces. [I. Zimmermann, Pharmazeutische Industrie, Springer-Verlag]. Whereas capillary forces caused by water vapour condensation can be controlled by suitable storage of the powders at reduced humidity, the Van-der-Waals forces and the electrostatic interactions between the (spray-dried) particles have proved a major challenge.
The interparticle interactions can be reduced by making the particle surface hydrophobic. This can be done by dissolving hydrophobic substances as additives with the protein or active substance and other suitable excipients and spray-drying them. The state of the art for rendering surfaces hydrophobic consists, inter alia, of the hydrophobic amino acid L-leucine (L. Mao et. al, 2004 Respiratory Drug Delivery IX, S. 653-656, A R. Najafabadi et al., 2004, Int J. Pharm. 2004 Nov. 5; 285(1-2):97-108). As only the surface coating is to be modified in this process, the amount of L-leucine needed is only 5-10 percent by weight (% w/w). Increasing the proportion of amino acid often leads to undesirable crystallisation effects, damaging the protein (Dissertation by Richard Fuhrherr, 2005, LMU Uni, Munich). The addition of other amino acids such as e.g. DL-asparagine, DL-arginine, DL-methionine, DL-phenylalanine and DL-tryptophan (N. Y. K. Chew et. al, 2002 Respiratory Drug Delivery VIII, S. 743-745) to the protein and preferably to the spray solution may have a beneficial effect on the aerodynamic characteristics of the particles. Besides the direct addition of the hydrophobic substance to the protein and particularly to the spray solution, the powder particles may be coated with additives in a further step. Substances which are particularly suitable for this are L-leucine, phospholipids and Mg-stearate (WO2004093848). Potential coating methods use gravity mixers, e.g. tumble mixers (US2005152849), but also mechanical mixing methods such as e.g. jet grinding (WO2004093848).
A conventional method of administering proteins and peptides is by parenteral administration. The active substance may for example be given intravenously, intramuscularly and subcutaneously. The state of the art is to administer the medicament through a cannula, e.g. combined with a syringe, a pen or as an infusion using an infusion bag. A disadvantage of this is that powder formulations have to be reconstituted in liquid before they are administered. Moreover, parenteral administration is not popular with patients because of needle phobia, a common complaint. For these reasons, parenteral treatments often have to be given by the doctor. By contrast, systemic inhaled formulations can be administered by the patients themselves.
Proteins/peptides can enter the bloodstream by passive diffusion or by active transportation through the lungs. In passive transportation, the absorption rate is a function of the size of the molecule of the active substance [J. S. Patton, Nature Biotechnology, 16, 141ff, 1998].
Whereas with small proteins such as insulin, for example, good bioavailablilities have been found (J. S. Patton, 1999 Advanced Drug Delivery Review, 35, 235-247) larger proteins and especially antibodies generally have a very low absorption rate. In order to develop an efficient form of medication, in spite of this, larger proteins have to be transported actively through the lung epithelium by specific mechanisms.
One possibility for actively transporting antibodies through the lung epithelium is the neonatal Fc-receptor (A. Bitonti, 2004, Respiratory Drug Delivery IX 0.79-85). It has been found that these receptors are present in sufficiently large numbers in the lungs not only of neonates but also in children and adults and can be used for actively transporting active substances.
When preparing powders containing protein for medical applications, particularly spray-dried powders or protein compositions, a particular challenge is to achieve, in addition to good protein stability, the most advantageous aerodynamic characteristics possible, so that the powders or the particles thereof, particularly spray-dried powders and particles, can penetrate deep into the lungs and thus easily enter the bloodstream.
In recent times more and more inhalable drugs have been developed (inhalable insulin as a development product made by Messrs Aradigm, Mannkind or Kos, K. Corkery, Respiratory Care, 45, 831ff, 2000) or are already on the market (e.g. Pulmozyme® as an inhaled form of recombinant human deoxyribonuclease I (rhDNase) or Exubera as an inhaled form of human insulin, cf. U.S. Pat. No. 5,997,848), for treating a variety of diseases. It has been found that certain drugs are easily absorbed in the lungs through the alveoli directly into the bloodstream. Administration by inhalation is particularly promising for administering macromolecules such as proteins, polypeptides and nucleic acids, which are difficult to administer by other routes (e.g. orally). This administration by inhalation may be effectively used both for systemic diseases and for local diseases of the lungs.
Pulmonary drug administration can be carried out by various methods, e.g. using liquid nebulizers, propellant-based inhalers (aerosol-based metered-dose inhalers=MDI), and dry powder dispersion devices. The development of propellant-based formulations is associated with a range of problems. Thus, the established chlorofluorocarbons (CFC's) can no longer be used, on account of their ozone-damaging properties. As a substitute alternative propellant gases may be used (HFA-143a/HFA227). The alternative propellant gases however often exhibit reduced solubility of the active substance, compared to the CFC's. In addition, the stability of the suspension is critical when preparing suspensions, with the result that further excipients are needed as mediators between the propellant gas and the particle. High dosage settings, such as are often needed antibodies, are difficult to achieve using MDI's. These factors have meant that MDI's have become more and more preferable for peptide and protein recipes. Dry powder dispersion devices, which are not dependent on propellant gas aerosol technology, are promising in the application of medicaments, which can easily be formulated as dry powders.
Many otherwise unstable macromolecules may be stabilised in the form of powders, particularly lyophilised or spray-dried powders, on their own or in conjunction with suitable excipients. However, the ability to administer pharmaceutical compositions as dry powders has its own problems. The metering of many pharmaceutical compositions is often critical. For this reason it is essential that every system for administering dry powder also administers the intended dose accurately, precisely and reliably in reality. This is not reliably ensured with the systems known hitherto. In addition, many drugs are very expensive. It is therefore important that the dry powder should be able to be delivered efficiently. It is also important that the powder is easily dispersible (capable of flight) before it is inhaled by the patient, so ensure adequate distribution and system absorption. These points are not ideally satisfied in the majority of conventional powders containing a protein or pharmaceutical active substance.
The problem therefore arises that in the powders used hitherto which contain a amount of protein, particularly spray-dried powders or protein compositions with pharmaceutical active substance, efficient and optimum pulmonary administration is not possible. Admittedly, it has been possible to achieve good protein stability in the powders used hitherto, but not optimum aerodynamic properties. For example large amounts of antibody in the powder, particularly in the spray-dried powder, causes severe clumping of the primary particles. These clumps are difficult to disperse, and this negatively affects the aerodynamic properties (doctoral thesis of Stefanie Schüle, Uni LMU 2005).
Thus the protein or pharmaceutical active substance which is to be administered has to be dosed in significantly larger amounts than are actually required, as, of the active substance used, only a fraction reaches the target site in the lungs. The danger of the side effects is also greater than when dosing is efficient.
The problem thus arises of providing alternative powders, particularly spray-dried powders or protein compositions, which in addition to having sufficient protein stability also have very good or improved aerodynamic properties.
A further aim of the invention is to provide corresponding alternative powders, particularly spray-dried powders or protein compositions, for use by inhalation, particularly for pharmaceutical or medical applications.
The problems on which the invention is based are solved by the following embodiments and by the objects and methods recited in the claims.